Trichomonas vaginalis is the etiologic agent of the most common non-viral sexually transmitted infection worldwide and is the most prevalent parasite found in the US population. Increased transmission of HIV in women chronically infected with this parasite and a recent increase in the incidence of drug resistant Trichomoniasis warrant a better understanding of the biology of this understudied pathogen. The proposed study aims to provide fundamental knowledge of the mechanisms underlying the regulation of gene expression in T. vaginalis. Our previous analyses of gene expression has led to series of observations that reveal distinct differences between the transcriptional machinery of this parasite and its human host that might be exploited for drug design. A promoter element, the intitator (Inr), which defines the start site of transcription and directs RNA polymerase II (RNAPII) to this site and a novel transcription factor (IBP39) that interacts with both the Inr and RNAPII has been discovered and characterized. We have also discovered the presence of introns in T. vaginalis genes and developed in vivo assays to characterize splicing in this primitive eukaryote. The specific aims of the proposed studies to continue this research are: (1) To characterize the interaction between IBP39 and the C-terminal domain (CTD) of RNA polymerase II (RNAPII), (2) To identify T. vaginalis proteins comprising the RNAPII pre-initiation complex (PIC) and the promoter motifs they interact with to coordinate initiation of transcription and (3) To define the structural properties and motifs that regulate RNA splicing in Trichomonas. These studies will reveal both conserved and divergent features of the basic machinery utilized to regulate gene expression in protists, Archaea, yeast and metazoa, providing insight into the evolution of gene regulation. Additionally, they will provide critical information on pivotal properties of gene expression that might lead to possible therapeutic applications.